Subtercola Vilae Sp. Nov., a Novel Actinobacterium from an Extremely High-Altitude Cold Volcano Lake in Chile
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by OceanRep Antonie van Leeuwenhoek https://doi.org/10.1007/s10482-017-0994-4 ORIGINAL PAPER Subtercola vilae sp. nov., a novel actinobacterium from an extremely high-altitude cold volcano lake in Chile Alvaro S. Villalobos . Jutta Wiese . Pablo Aguilar . Cristina Dorador . Johannes F. Imhoff Received: 23 August 2017 / Accepted: 1 December 2017 Ó The Author(s) 2017. This article is an open access publication Abstract A novel actinobacterium, strain DB165T, between the amino acids in its peptidoglycan is type was isolated from cold waters of Llullaillaco Volcano B2c; 2,4-diaminobutyric acid is the diagnostic dia- Lake (6170 m asl) in Chile. Phylogenetic analysis mino acid; the major respiratory quinones are MK-9 based on 16S rRNA gene sequences identified strain and MK-10; and the polar lipids consist of phos- DB165T as belonging to the genus Subtercola in the phatidylglycerol, diphosphatidylglycerol, 5 glycol- family Microbacteriaceae, sharing 97.4% of sequence ipids, 2 phospholipids and 5 additional polar lipids. similarity with Subtercola frigoramans DSM 13057T, The fatty acid profile of DB165T (5% [) contains iso- 96.7% with Subtercola lobariae DSM 103962T, and C14:0, iso-C16:0, anteiso-C15:0, anteiso-C17:0, and 96.1% with Subtercola boreus DSM 13056T. The cells the dimethylacetal iso-C16:0 DMA. The genomic were observed to be Gram-positive, form rods with DNA G?C content of strain DB165T was determined irregular morphology, and to grow best at 10–15 °C, to be 65 mol%. Based on the phylogenetic, pheno- pH 7 and in the absence of NaCl. The cross-linkage typic, and chemotaxonomic analyses presented in this A1 A. S. Villalobos Á J. Wiese Á J. F. Imhoff (&) A16 P. Aguilar A2 Marine Microbiology, GEOMAR Helmholtz Centre for A17 Lake and Glacier Ecology Research Group, Institute of A3 Ocean Research Kiel, Du¨sternbrooker Weg 20, A18 Ecology, University of Innsbruck, Techniker Str. 25, A4 24105 Kiel, Germany A19 6020 Innsbruck, Austria A5 e-mail: [email protected] A20 C. Dorador A6 A. S. Villalobos A21 Centre for Biotechnology and Bioengineering (CeBiB), A7 e-mail: [email protected] A22 Universidad de Antofagasta, Antofagasta, Chile A23 e-mail: [email protected] A8 J. Wiese A9 e-mail: [email protected] A10 P. Aguilar Á C. Dorador A11 Laboratorio de Complejidad Microbiana y Ecologı´a A12 Funcional and Departamento de Biotecnologı´a, Facultad A13 de Ciencias del Mar y Recursos Biolo´gicos, Universidad A14 de Antofagasta, Antofagasta, Chile A15 e-mail: [email protected] 123 Antonie van Leeuwenhoek study, strain DB165T (= DSM 105013T = JCM plates. Stock cultures were maintained in SGG 32044T) represents a new species in the genus medium containing 10 g starch, 10 g glucose, 10 ml Subtercola, for which the name Subtercola vilae sp. glycerol (99.7% v/v), 5 g soy peptone, 2.5 g corn steep nov. is proposed. solids, 2 g yeast extract, 3 g CaCO3, 1 g NaCl, and 18 g agar in 1 l deionised water (Goodfellow and Keywords Cold environments Á Llullaillaco Fiedler 2010). DB165T was cryopreserved using volcano Á Microbacteriaceae Á New species Á CRYOBANK (Mast Diagnostica GmbH, Germany) Subtercola vilae for long term storage at - 80 °C. Gram-staining was prepared using the Color Gram 2 kit (BioMe´rieux, France), following the manufac- turer’s protocol. Endospore staining was performed Introduction using the green malaquite method and light micro- scopy (Schaeffer and Fulton 1933). Cell morphology, Members of the family Microbacteriaceae are widely shape and size were determined using scanning distributed in terrestrial and aquatic environments or electron microscopy (SEM) according to Ga¨rtner associated with macroorganisms (Evtushenko 2012). et al. (2008), after cultivation of trypticase soy Some representatives, including species of the genus medium (Trypticase Soy Broth (Becton, Dickinson Subtercola, have been found in cold environments and company, France) supplemented with 18 g agar such as glacial ice (Christner et al. 2007), boreal l-1. groundwater (Ma¨nnisto¨ et al. 2000), and Antarctic The reference strains S. boreus DSM 13057T and S. sediments (Li et al. 2010). At present the genus frigoramans DSM 13057T were obtained from the Subtercola contains three validly named species, German Collection of Microorganisms and Cell Subtercola boreus, Subtercola frigoramans and Cultures (DSMZ) and cultured under the same condi- Subtercola lobariae; the first two were isolated from tions as strain DB165T for comparative purposes. Finnish groundwater (Ma¨nnisto¨ et al. 2000) and the third from the lichen Lobaria retigera (Si et al. 2017). Physiological characteristics Based on the high similarity of 16S rRNA gene sequences ([ 96%), other isolates from cold habitats, Enzyme activities and utilisation of carbon sources for such as Antarctic and Artic waters as well as glaciers strain DB165T, S. boreus DSM 13057T and S. were found to be affiliated to Subtercola (Singh et al. frigoramans DSM 13057T were examined using API 2014; Zhang et al. 2013; Peeters et al. 2011). ZYM, API 20E and API 50CH (BioMe´rieux, France), In this study, we characterise strain DB165T, following the manufacturer’s recommendations. The isolated from a water sample of Llullaillaco Volcano effect of sodium chloride (0, 0.1, 0.3, 0.6, 0.9, 1, 2.5, 5, Lake (6170 m) in Chile, one of the highest-elevation 7.5, and 10% w/v) and pH (2, 3, 4, 5, 6, 7, 8, 9 and 10) lakes on Earth. According to its distinct properties, on the growth was tested according to Kutzner (1981), strain DB165T is proposed as the type strain of the new using ISP2 medium containing 4 g yeast extract, 10 g species Subtercola vilae. malt extract, 4 g dextrose, and 18 g agar in 1 l of distilled water. The optimal range of temperature was tested at 5, 10, 15, 20, 28 and 30 °C using SGG Materials and methods medium. Isolation and cell morphology Chemotaxonomic analyses Strain DB165T was obtained from a water sample Polar lipids were extracted according to a modified collected at the Llullaillaco volcano lake protocol of Bligh and Dyer (1959), and the total lipid (S24°42.8780, W68°33.3100) on 18 January 2013, material was detected using molybdatophosphoric using R2A medium (DIFCO) supplemented with acid and specific functional groups were detected 18 g agar l-1. Pure cultures were obtained after three using spray reagents specific for defined functional successive transfers of single colonies to R2A medium groups (Tindall et al. 2007). The lipoquinones were 123 Antonie van Leeuwenhoek extracted and identified using the two-stage method Results described by Tindall (1990a, b). After cultivation at 25 °C, fatty acid methyl esters were obtained by Morphological and physiological characteristics saponification, methylation and extraction using minor modifications of the method of Miller (1982) Colonies of strain DB165T were observed to be sticky, and Kuykendall et al. (1988) The fatty acid methyl golden yellow after growth at the optimal growth esters mixtures were separated and identified using the temperature of 10–15 °C for 6–7 days (also after Sherlock Microbial Identification System (MIS) 7–10 days at 28 °C), but are pale yellow after growth (MIDI, Microbial ID, Newark, DE 19711, USA). at 5 °C for 2–3 weeks. Optimum growth is observed at The peptidoglycan was obtained from 4 g wet 10–15 °C (range from 5 to 28 °C). No growth occurs weight cell pellet according to the method of Schleifer at 30 °C. Strain DB165T tolerates only low concen- (1985). The peptidoglycan analyses were performed trations (up to 0.9%) of NaCl and grows best in the according to Schumann (2011). absence of NaCl. The pH range for growth is from 5 to Analyses of polar lipids, respiratory quinones, 8, with an optimum at pH 7. Cells show no motility and whole-cell fatty acids and peptidoglycan analyses form no spores, they are irregular short rods of 0.5 lm were carried out by the Identification Service of the width and 1.0–1.2 lm length, Gram-positive and have DSMZ—Deutsche Sammlung von Mikroorganismen an irregular shape as seen under SEM. Some of the und Zellkulturen GmbH (Braunschweig, Germany). cells are thicker at the ends. Occasionally, coccoid cells were observed (Fig. 1). Variable cell shapes have DNA base composition also been reported for S. boreus and S. frigoramans (Ma¨nnisto¨ et al. 2000). DNA was extracted using the DNeasy Blood & Tissue The metabolic properties of strain DB165T,in kit (QIAGEN). The G?C content was calculated from comparison with the type strains of S. boreus and S. the genome sequence, which was determined with frigoramans are shown in Table 1. Strain DB165T was Nextseq 500 (Illumina). The quality of the sequences found to metabolise inositol, D-sorbitol, D-sucrose, D- was checked and filtrated using Trimmomatic (adap- melibiose, glycerol, L-arabinose, D-xylose, methyl-b- ters, [ Q30, [ 1000 bp) (Bolger et al. 2014). The D-xylopyranoside, D-galactose, D-glucose, D-fructose, genome was assembled using SPAdes (Kmer = 121) D-mannose, L-rhamnose, D-mannitol, amygdalin, (Bankevich et al. 2012). Phylogenetic analyses DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN) with modifications. The 16S rRNA gene sequence was amplified by PCR using PureTaq Ready-To-Go PCR beads (GE Healthcare) and sequencing according to Ga¨rtner et al. (2008). The 16S rRNA gene sequence of strain DB165T was aligned with sequences of 22 selected type strains of the family Microbacteriaceae, including members of the genera Subtercola, Frondihabitans and Agreia, and in addition Cellulomonas carbonis KCTC 19824T as outgroup using SINA (Pruesse et al. 2012). Phylogenetic trees were constructed using the neigh- bour-joining (Saitou and Nei 1987) and maximum- likelihood algorithms using MEGA version 6.0 (Ta- mura et al.